Multisignal reagents for labeling analytes

ABSTRACT

Provided is a composition comprising an analyte bound covalently or through a first binding pair to a polymer. In this composition, the analyte is less than about 2000 MW; the polymer further comprises more than one signal or first member of a second binding pair; and the analyte is not a member of the first binding pair or the second binding pair. Also provided is an assay for an analyte. The assay comprises: combining a sample suspected of containing the analyte with the above-described composition and a binding agent that binds to the analyte; and detecting the signal or the first member of the second binding pair that is bound to the binding agent. Additionally provided is a multisignal labeling reagent comprising a first polymer covalently bound to (a) a reactive group or a first member of a first binding pair, and (b) more than one digoxigenin molecule.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 15/609,380,filed May 31, 2017, which is a continuation-in-part of U.S. patentapplication Ser. No. 13/065,101, filed Mar. 14, 2011, now U.S. Pat. No.9,156,986, which is a continuation-in-part application of U.S. patentapplication Ser. No. 12/399,393, filed Mar. 6, 2009, now U.S. Pat. No.8,394,949, which is a divisional of application Ser. No. 10/407,818,filed Apr. 3, 2003, now U.S. Pat. No. 7,514,551, the contents of each ishereby incorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 27, 2018, isnamed ENZ-65-CIP2-D1-CON-103.txt and is 1.539 bytes in size.

BACKGROUND OF THE INVENTION (1) Field of the Invention

The present application generally relates to assays for detectinganalytes, e.g., proteins and small molecules (haptens). Morespecifically, this application provides reagents for improving thedetection and quantitation of an analytes with assays that utilize anagent, such as an antibody, that binds to the hapten.

(2) Description of the Related Art

There is a high demand to determine the presence and concentration ofanalytes of interest, for example protein and small molecule (“hapten”)analytes, which can be of environmental or medical concern. Examples ofhapten analytes include fungal or microbial toxins as a threat to foodsafety, and drugs, steroids, hormones, proteins, peptides, lipids,sugars, receptors, nucleic acids, vitamins, etc., e.g., in mammalianfluid or tissue samples, to identify an intoxication, control themedication of therapeutic drugs with a narrow therapeutic window, etc.

Assays (e.g., immunoassays) utilizing an agent that binds to an analyteof interest (e.g., an antibody) have been developed for detectingnumerous analyte compounds at very low levels. As is known in the art,antibodies that bind to small molecule analytes can be developed by, forexample, using phage display techniques, or by utilizing an immunogenthat comprises the analyte, or an analog of the analyte, covalentlyconjugated to a carrier protein or other immunogenic macromolecule. Whenusing hapten immunogens, antibodies are generally elicited to theportion of the analyte that is distal to the chemical bond through whichthe hapten is conjugated to the carrier, such that the distal portionbecomes an epitope of the carrier-hapten complex.

Immunoassays for analytes often take the form of competitive bindingassays, where, for example, a labeled analyte competes with analytes inthe sample for binding to antibodies fixed on a solid matrix. In thoseassays, the signal from the label decreases with increasingconcentration of analyte in the sample. Examples of such an assayincludes known assays for cAMP and vitamin D (see, e.g., Enzo LifeSciences, “cAMP Complete ELISA kit”; GE Life Sciences, “Cyclic-AMP AssayKit”; Caymen Chemical Company, “Cyclic AMP EIA Kit”; PerkinElmer,“AlphaScreen® cAMP Assay Kit”; and Promega, “GloSensor™ cAMP Assay”).These competitive assays and similar assays are often improved byimproving the signal intensity of the label.

The use of non-radioactive labels in biochemistry and molecular biologyhas grown exponentially in recent years. Among the various compoundsused as non-radioactive labels, aromatic dyes that produce a fluorescentor luminescent signal are especially useful. Notable examples of suchcompounds include fluorescein, rhodamine, coumarin and cyanine dyes suchas Cy3 and Cy5. Composite dyes have also been synthesized by fusing twodifferent dyes together. See, e.g., Lee et al., 1992; and U.S. Pat. Nos.5,945,526 and 6,008,373.

Non-radioactive labeling methods have been developed to attachsignal-generating groups onto proteins, nucleic acids and haptens. Thisis generally achieved by modifying labels with chemical groups such thatthey would be capable of reacting with, e.g., the amine, thiol, andhydroxyl groups on proteins or haptens. Examples of reactive groupsutilized for this purpose include activated esters such asN-hydroxysuccinimide esters, isothiocyanates and other compounds.

Labeled nucleotides are used for the synthesis of DNA and RNA probes inmany enzymatic methods including terminal transferase labeling, nicktranslation, random priming, reverse transcription, RNA transcriptionand primer extension. Labeled phosphoramidite versions of thesenucleotides have also been used with automated synthesizers to preparelabeled oligonucleotides. The resulting labeled probes are widely usedin such standard procedures as northern blotting, Southern blotting, insitu hybridization, RNase protection assays, DNA sequencing reactions,DNA and RNA microarray analysis and chromosome painting.

There is an extensive literature on chemical modification of nucleicacids by means of which a signal moiety is directly or indirectlyattached to a nucleic acid. See, e.g., U.S. Pat. Nos. 4,711,955 and5,241,060, 4,952,685, 5,013,831, 7,166,478 and 7,514,551, and U.S.Patent Publication 2011/0318788.

The presence and nature of a linker arm may also improve the signalingcharacteristics of the labeled target molecule (see, e.g., U.S. PatentPublication 2011/0218788 and U.S. Pat. No. 7,514,551).

BRIEF SUMMARY OF THE INVENTION

The invention provided herein is based in part on the discovery that amultisignal labeling reagent, e.g., as described in U.S. PatentPublication 2011/0318788 and U.S. Pat. No. 7,514,551, can beadvantageously utilized for labeling analytes, e.g., protein or on smallmolecule analytes, for example as a reagent in competitive immunoassays.

Thus, in some embodiments, a composition comprising an analyte boundcovalently or through a first binding pair to a polymer is provided. Inthese embodiments, the analyte is less than about 2000 MW; the polymerfurther comprises more than one signal or first member of a secondbinding pair; and the analyte is not a member of the first binding pairor the second binding pair.

In other embodiments, an assay for an analyte is provided. The assaycomprises: (a) combining a sample suspected of containing the analytewith a detection reagent and a binding agent that binds to the analyte,wherein the detection reagent comprises the analyte or an analyte analogbound covalently or through a first binding pair to a polymer, saidpolymer further comprising more than one signal or first member of asecond binding pair, wherein the analyte or analyte analog is not amember of the first binding pair or the second binding pair; (b)removing any of the detection reagent that is not bound to the bindingagent; and (c) detecting the signal or the first member of the secondbinding pair that is bound to the binding agent. In these embodiments,the amount of the signal or the first member of the second binding pairbound to the binding agent is inversely proportional to the analyte inthe sample.

Additionally provided is a multisignal labeling reagent comprising afirst polymer covalently bound to (a) a reactive group or a first memberof a first binding pair, and (b) more than one digoxigenin molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the results of an immunoassay for cAMPutilizing a composition of the present invention.

FIG. 2 is a graph showing the results of a comparison of four labelingreagents.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Additionally, the use of “or” is intended to include“and/or”, unless the context clearly indicates otherwise.

The present invention is based in part on the discovery that amultisignal labeling reagent, e.g., as described in U.S. PatentPublication 2011/0318788 and U.S. Pat. No. 7,514,551, can beadvantageously utilized on small molecules, for example as a reagent incompetitive immunoassays.

Thus, in some embodiments, a composition comprising an analyte boundcovalently or through a first binding pair to a polymer is provided. Inthese embodiments, the analyte is less than about 2000 MW; the polymerfurther comprises more than one signal or first member of a secondbinding pair; and the analyte is not a member of the first binding pairor the second binding pair.

In various aspects of these embodiments, the analyte is less than about1000 MW, less than about 500 MW, less than about 250 MW, less than about200 MW, or less than about 150 MW.

As used herein, a polymer is an organic molecule comprising at least 3repeating monomeric units such as amino acids, sugars, nucleotides ornucleotide analogs. The polymer of the composition can be comprised ofany form of organic monomer and can be less than about 10 monomers, lessthan about 20 monomers, less than about 50 monomers, less than about 100monomers, or about 100 or more monomers.

Non-limiting examples of polymers made from such monomeric units includenucleic acids, abasic nucleic acids, peptide nucleic acids,polypeptides, proteins, oligosaccharides, polysaccharides and organicpolymers. The polymers used in the present invention may be isolatedfrom biological sources or they may be created synthetically or invitro. The polymers may also comprise multiples of only one particulartype of monomeric unit, or they may comprise different types ofmonomeric units. For example, a chimeric oligomer or polymer can be anucleic acid construct that comprises both a normal nucleic acid segmentand a peptide nucleic acid segment, a combination of nucleotides andamino acids, or a combination of a segment of an abasic nucleic acid anda segment comprising a peptide nucleic acid.

Additionally, even when the monomeric units of the polymer are the sametype of compound (e.g., all deoxyribonucleotides), they may be the sameor they may be different. For instance, a nucleic acid polymer may be ahomopolymer comprising a reiteration of a single base or it can be aheteropolymer having varied nucleotides. A polypeptide polymer may behomopolymeric and comprise multiples of a single amino acid or it may beheteropolymeric and comprise different amino acids. The labels in anoligomeric or polymeric labeling reagent may also be the same or theymay be different. For instance, a labeling reagent that comprises twodifferent dyes attached at discrete intervals on a polynucleotide mayparticipate in energy transfer for signal generation.

The polymers in these compositions may comprise a single chain structurelinking the monomeric units together or they may comprise more than onechain. For example, branched, double-stranded and triple-strandednucleic acids may all find use with present invention. Additionally, thepolymer comprising the signals or the first members of a second bindingpair can be hybridized to the polymer that is bound to the analyte. Suchmulti-chain structures may provide useful properties. For example, adouble-stranded nucleic acid is more rigid than a single strandednucleic acid. The use of a double-stranded structure may allow bettercontrol over the distribution or spacing of labeled moieties whereproximity or lack of proximity may be desirable. As is known, efficientsignal generation by means of energy transfer depends upon a closeproximity of donor and acceptor moieties and as such, establishment of aproximity between these moieties can be beneficial. Additionally, if asingle dye species is being used as signal generators, a close proximityof some dye molecules can lead to a self-quenching phenomenon, andspreading out the locations of the dyes could thus be beneficial. Theuse of more than one chain may also convey other useful properties suchas increasing the amount of signal generated or increasing the chargenumber. Multiple chains may also endow the system with flexibility ofuse. For example, a first nucleic acid strand may comprise a reactivegroup and a second nucleic acid strand with complementary sequences cancomprise signal groups. By complementary base pairing between thesestrands, a complex can be formed that comprises a reactive group andsignaling groups. See, e.g., FIG. 1 of U.S. Pat. No. 7,514,551. Forexample, they may comprise termini or extended chains with extendedmultiple charged groups. Other groups that may offer useful additionalproperties may also find use with the present invention.

In some embodiments, the polymer is an oligopeptide. The oligopeptidepolymer can be more than about 100 amino acids or amino acid analogs, orless than about 100 amino acids or amino acid analogs, e.g., less thanabout 90, 80, 70, 60, 50, 40, 30, 20, 10 or 5 amino acids or amino acidanalogs. In other embodiments, the polymer is a nucleic acid. Such apolymer can comprise any known nucleotide or nucleotide analog known inthe art. The nucleic acid polymer can be more than about 100 nucleotidesor nucleotide analogs, or less than about 100 nucleotides or nucleotidesanalogs, e.g., less than about 90, 80, 70, 60, 50, 40, 30, 25, 20, 15,10 or 5 nucleotides or nucleotide analogs. The nucleic acid polymers canbe synthesized by any means known in the art, for example as describedin U.S. Patent Publication 2011/0318788 and U.S. Pat. No. 7,514,551.

In the invention compositions, the polymer can be bound to the analyteeither covalently or through a first binding pair. Where the analyte iscovalently bound to the polymer, the analyte and polymer can be joinedby any method known in the art for the particular analyte and polymer.See, e.g., Example 1. As is known in the art, the analyte (or thepolymer) can be modified to comprise a reactive group that reacts with amoiety on the polymer (or analyte). Where an immunoassay is alreadyavailable for the analyte, the methods utilized to conjugate the analyteto the carrier protein to prepare the immunogen can generally also beutilized to conjugate the analyte to the polymer. Examples of reactivegroups include but are not limited to active esters, groups capable offorming a carbon-carbon bonds and groups capable of forming bonds with0, N or S. Specific examples of such groups are isothiocyanate,isocyanate, monochlorotriazine, dichlorotriazine, mono- or di-halogensubstituted pyridine, mono- or di-halogen substituted diazine,maleimide, aziridine, sulfonyl halogen substituted diazine, maleimide,aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester,hydroxysulfosuccinimide ester, imido ester, hydrazine, azidonitrophenyl,azide, 3-(2-pyridyl dithio)-proprionamide, glyoxal, aldehyde,carbon-carbon double bonds, mercury salts, and any group capable ofreacting with carbon-carbon double bonds, amines, hydroxyl groups,sulfhydryl groups and halogens.

Where the analyte is bound to the polymer through a first binding pair,the analyte is conjugated to one member of the first binding pair andthe polymer is conjugated to the other member of the first binding pair.Non-limiting examples of binding pairs are a sugar-lectin, anantigen-antibody, a ligand-ligand receptor, a hormone-hormone receptor,an enzyme-substrate, a biotin-avidin, and a biotin-streptavidin. Methodsfor conjugating haptens and polymers to various members of binding pairsare well-known in the art. For the purposes of the inventioncompositions, a DNA supramolecular binding molecule or two DNAsupramolecular binding molecules covalently joined by a linker group, asdescribed in U.S. Patent Publication 2011/0318788, is considered amember of a binding pair, where the nucleic acid to which the DNAsupramolecular binding molecule(s) bind is considered both the polymerand the other member of the binding pair.

As used herein, a “linker” or “linker arm” is a chemical moiety thatseparates one component of the composition (e.g., the polymer, hapten,binding pair member or signal) from another component. Thus, a linkermay be used for example to separate the hapten from the first bindingpair, the hapten from the polymer, the polymer from the first bindingpair, the polymer from the second binding pair, the polymer from thesignal, or the second binding pair from the signal.

A linker comprises a chain of atoms of any length that may be comprisedof carbon, nitrogen, oxygen, sulfur in any combination and any otherpossible atom. The connecting chain can be saturated, unsaturated or cancontain aromatic rings and the linking chain can be flexible or rigid.The connecting chain can further comprise any of the rigid unitsdisclosed, e.g., in U.S. Patent Publication 2005/0137388.

The linker in these compounds can be rigid or flexible. Rigid linkershave been utilized with dimer intercalators. See, e.g., Glover et al.(2003). However, flexible linkers do not require the precise designrequired of rigid linkers, where the linker must precisely separate andorient the DNA supramolecular binding molecules to properly insert intothe nucleic acid.

In some embodiments, the linker comprises an unsubstituted C₁-C₂₀straight-chain, branched or cyclic alkyl, alkenyl or alkynyl group, asubstituted C₁-C₁₀ straight-chain, branched or cyclic alkyl, alkenyl oralkynyl group wherein one or more C, CH or CH₂ groups are substitutedwith an O atom, N atom, S atom, NH group, CO group or OCO group, or anunsubstituted or substituted aromatic group. In more specificembodiments, the linker is (CH₂)1-10-NH—(CH₂)1-10-. In still morespecific embodiments, the linker is —(CH₂)₁₋₅—NH—(CH₂)₁₋₅—. One usefullinker within these embodiments is —(CH₂)₃—NH—(CH₂)₄— (spermidine—seeExamples 20-22 of U.S. Patent Publication 2011/0318788).

Linkers can be covalently attached to any of the substituents of theinstant compositions by any means known in the art, e.g., through areactive group as discussed above.

In some embodiments, the polymer is covalently bound to the signalmoieties either directly or through a linker. Non-limiting examples ofsignals useful for these compositions are fluorescent dyes, coloreddyes, radioactive molecules, chemiluminescent molecules and enzymes.Where the signal is an enzyme, the enzyme is capable of modifying asubstrate to create a detectable signal. Non-limiting examples of suchenzymes include alkaline phosphatase, horseradish peroxidase andluciferase.

In other embodiments, the polymer is covalently bound to more than onefirst member of a second binding pair, e.g., digoxigenin, fluorescein,biotin or dinitrophenol. In these embodiments, the first member of thesecond binding pair can be made into a detectable signal by adding asignal molecule that is bound to the second member of the second bindingpair. Thus, a polymer bound to a hapten (e.g., cAMP—see Examples) thatfurther comprises, e.g., more than one biotin moiety can be utilized inan assay where the biotin moieties are detected by addingstreptavidin-alkaline phosphatase then an alkaline phosphatasesubstrate. See Example 3 below. In some of these embodiments, thecomposition further comprises more than one signal covalently bound to asecond member of the second binding pair, wherein each signal-secondmember of the second binding pair is noncovalently bound to each firstmember of the second binding pair.

The compositions of these embodiments may also contain additional alkyl,aryl and/or polar or charged groups on any component of the composition,e.g., the hapten, the polymer, a binding pair member, a linker, or thesignal. The polar or charged groups may include but are not limited tohalogen, substituted or unsubstituted alkyl or aryl groups, saturated orunsaturated alkyl groups, alkoxy, phenoxy, amino, amido, and carboxylgroups, polar groups such as nitrates, sulfonates, sulfhydryl groups,nitrites, carboxylic acids, phosphates or any other such group orsubstituent.

The analytes for these compositions can be any hapten that can bemanipulated as described above. Useful analytes include mammaliancellular metabolites, metabolites of microorganisms, medications,illicit drugs, vitamins, environmental pollutants, pesticides, andtoxins. Specific non-limiting examples include cAMP, cGMP,8-bromoadenosine 3′,5′-cyclic monophosphate, cholesterol, ahydroxycholesterol, vitamin D, 25-hydroxy vitamin D, vitamin B12,vitamin E, vitamin B1, vitamin B6, ascorbic acid, retinol, biotin,folate, legumin, salbutamol, melamine, sulfaquinoxaline, an inositolphosphate, a phosphatidyl inositol phosphate, prednisone, pregnenolone,dexamethasone, triamcinolone, fludrocortisone, dihydrotachysterol,oxandrolone, testosterone, dihydrotestosterone, nandrolone,norethindrone, medroxyprogesterone acetate, progesterone, aglucocorticoid, aldosterone, estrogen, oxytocin, androstanediolglucuronide, bilirubin, warfarin, an estrogen, methotrexate, tobramycin,acetaminophen, encainide, fluoxetine, gentamycin, an aminoglycoside,amikacin, coenzyme Q10, theophylline, phenytoin, cimetidine, disulfiram,trazodone, ethanol, halothane, phenylbutazone, azapropazone, ibuprofen,amiodarone, imipramine, miconazole, metronidazole, nifedipine,chloramphenicol, trimethoprim, a sulfonamide, rifampin, cisplatin,vinblastine, bleomycin, oxacillin, nitrofurantoin, phenobarbital,primidone, carbamazepine, metoclopramide, cholestyramine, colestipol,neomycin, sulfasalazine, digoxin, indomethacin, diltiazem, erythromycin,tetracycline, itraconazole, nicardipine, triamterene, spironolactone,chlorpromazine, a cyclosporin, nortriptyline, ethosuximide, imipramine,codeine, lorazepam, topiramate, disopyramide, levetiracetam, clobazam,oxcarbazepine, fructosamine, caffeine, methaqualone, meprobamate,fluphenazine, a barbiturate, a phenothiazine, serotonin, valproic acid,digitoxin, maprotiline, lidocaine, mexiletine, primidone, risperidone,OH-rispiridone, a porphyrin, haloperidol, flecainide, tocainide,acetazolamide, a sulfonamide, verapamil, a metanephrine, an oxidizednucleotide, a mycotoxin, tetrahydrocannabinol, a cannabinoid, cocaine,LSD, an amphetamine, a barbiturate, heroin, methadone, nicotine,cotinine, a benzodiazepine, bis(2-ethylhexyl) phthalate, bisphenol A,hydralazine, atrazine, an organochloride insecticide, an organophosphateinsecticide, or a carbamate insecticide. In some embodiments, theanalyte is a steroid hormone, a glucocorticoid, oxytocin, cAMP, cGMP,hydroxycholesterol, vitamin D or 25-hydroxy vitamin D. In thesecompositions, the analyte could also be an analog of a compound ofinterest, e.g., any of the above compounds, particularly where theanalog is able to compete with sample analyte for binding sites to abinding agent (e.g., an antibody), thus making the analog-polymercomposition useful for competitive assays for the analyte (see furtherdiscussion below).

It is noted that certain analytes known to be members of a binding paircan also usefully be utilized as analytes in the invention compositions.An example is digoxigenin as an analyte, e.g., where the polymer isattached to the digoxigenin analyte through a biotin-streptavidinbinding pair. In such a case, the digoxigenin is considered an analyteand not a member of a binding pair.

The above-described compositions, as well as multisignal labelingreagents bound to larger analytes such as proteins (for example asdescribed in U.S. Patent Publication 2011/0318788 and U.S. Pat. No.7,514,551) are particularly useful in assays for identification orquantitation of the analyte in a sample. The assays utilize a bindingagent to the analyte, e.g., an antibody or analyte-binding fragmentthereof.

Thus, an assay for an analyte is provided. The assay comprises: (a)combining a sample suspected of containing the analyte with a detectionreagent and a binding agent that binds to the analyte, wherein thedetection reagent comprises the analyte or an analyte analog boundcovalently or through a first binding pair to a polymer, said polymerfurther comprising more than one signal or first member of a secondbinding pair, wherein the analyte or analyte analog is not a member ofthe first binding pair or the second binding pair; and (b) detecting thesignal or the first member of the second binding pair that is bound tothe binding agent. In these embodiments, the amount of the signal or thefirst member of the second binding pair bound to the binding agent isinversely proportional to the analyte in the sample.

The antibodies useful for these assays can be immunoglobulins of anyvertebrate species, e.g., rabbit, goat, mouse, sheep, chicken, etc. andcan be polyclonal or monoclonal. They can include the Fc region or theycan be Fab or Fab2 fragments or otherwise engineered or manipulated toexclude all or part of the Fc region. Additionally, they can be from anysource, e.g., from the serum of an animal injected with an immunogensuch as any of the immunogens described above, or they can be fromculture or ascites as is known in the art of hybridoma technology.Alternatively, they can be from recombinant sources, e.g., as describedin Winter et al., 1994, or Charlton and Porter, 2002.

Any immunoassay known in the art as useful for analyte detection can beutilized for the instant assays. The relevant immunoassays generallyutilize a competitive format, i.e., where the analyte in the samplecompetes with a labeled analyte or labeled hapten or hapten analog(“detection reagent”) for anti-analyte antibody binding sites such thatless detection reagent is bound when there is more hapten in the sample.Thus, in these competitive assays, an increasing amount of analyte inthe sample results in less detection reagent bound to the solid phase,and consequently less signal. In various competitive assays, the samplecan be added with the detection reagent to compete directly for antibodybinding sites, or the sample and detection reagent can be addedsequentially such that the detection reagent simply binds where thesample hapten is not bound.

The competitive assay for these embodiments can be homogeneous, wherethe signal detection step is performed without removing detectionreagent that is not bound to the reagent. Nonlimiting examples includeassays that utilize a change in FRET from a fluorescent signal when thedetection reagent is bound to the binding agent. Alternatively, theassay can be heterogeneous, where detection reagent that is not bound tothe binding agent is removed, so that the only signal is from bounddetection reagent.

In some embodiments, the immunoassay is a direct competitive assay,utilized where the detection reagent comprises the analyte bound to thepolymer comprising the signal, or an indirect competitive immunoassay,where the detection reagent comprises a first member of a second bindingpair, e.g., a second antibody (for example an anti-digoxigeninantibody), biotin, or streptavidin. In the latter case, the amount ofbound detection reagent is determined by adding the second member of thesecond binding pair that is bound to a signal (e.g., adigoxigenin-fluorescent dye complex, a biotin-alkaline phosphatasecomplex, a streptavidin-luciferase complex, etc.).

The immunoassays provided herein can take any format known in the art.In some embodiments, analyte antibodies are bound to the solid phase,either directly or indirectly, the latter being where the solid phase iscoated with an anti-antibody (for example goat antibodies that bind torabbit IgG antibodies [goat anti-rabbit IgG]) and the analyte antibodiesare bound to the anti-antibody. The anti-antibodies are also known as“second antibodies.” In these assays, the sample and detection reagentis added to the solid phase to compete with antibody binding sites onthe coated solid phase. After washing, the signal is generated, whichmeasures the amount of detection reagent that is bound to the solidphase. Numerous particular assays with this configuration can be devisedwithout undue experimentation.

Numerous specific immunoassay formats are known that could be utilizedwith analyte-multisignal labeling reagents to determine an analyte in asample. See, e.g., U.S. Patent Publication 2012/0115169, which providesa description of various relevant immunoassays. As described therein,the assay is performed in a liquid phase or on a solid phase, e.g., on abead or a microplate, for example a 96 well microtiter plate.Nonlimiting examples of immunoassays useful in these methods are aradioimmunoassay, a Luminex® assay (see, e.g., Wong et al., 2008), amicroarray assay, a fluorescence polarization immunoassay (see, e.g.,U.S. Pat. No. 4,585,862), an immunoassay comprising a Förster resonanceenergy transfer (FRET) signaling system (see, e.g., Blomberg et al.,1999; Mayilo et al., 2009), a scintillation proximity assay, afluorescence polarization assay, a homogeneous time-resolvedfluorescence assay, an amplified luminescence assay (“ALPHAScreen”), anenzyme complementation assay, an electrochemiluminescence assay, and anenzyme immunoassay (a.k.a. enzyme linked immunosorbent assay [ELISA]).As is well known in the art, in ELISA, an enzyme combined with asubstrate that becomes colored upon reaction with the enzyme providesthe signal to quantify the antigen in the sample. See, e.g., O'Beirneand Cooper, 1979.

In various embodiments of these assays, the binding agent is an antibodyor analyte-binding fragment thereof. In additional embodiments, thebinding agent is bound to a solid phase.

In some embodiments, the analyte is less than about 2000 MW. In theseembodiments, the detection reagent can be any of the analyte-polymercompositions described above. In some embodiments, the detection reagentcomprises an analog to the analyte of interest bound to the polymer.Such a configuration may be favored to provide for more convenientproduction of the detection reagent, where the analyte analog providesfor easier chemistry for conjugation to the polymer or first bindingpair member. Utilization of an analyte analog in the detection reagentcan also provide for more favorable conditions for binding to thebinding agent, for example where a detection reagent with an analyteanalog competes with sample analyte for antibody binding sites morefavorably (e.g., providing better sensitivity or less background) than adetection reagent that utilizes the analyte.

In some aspects of these embodiments, the polymer comprises (a) morethan one signal or (b) more than one first member of a second bindingpair and a signal covalently bound to a second member of the secondbinding pair. In these aspects, each signal-second member of the secondbinding pair is noncovalently bound to each first member of the secondbinding pair. In alternative aspects, the signal covalently bound to asecond member of the second binding pair is added after the combiningstep or the removing step.

With any of the assays described above, the signal can be any detectablelabel known in the art, for example a fluorescent dye, a colored dye, aradioactive molecule, a chemiluminescent molecule or an enzymes.Examples of useful enzymes in these embodiments are horseradishperoxidase, alkaline phosphatase and luciferase.

These assays can be used to detect any analyte that has a cognatebinding agent or to which a binding agent (e.g., an antibody) can besynthesized. Useful analytes include mammalian cellular metabolites,metabolites of microorganisms, medications, illicit drugs, vitamins,environmental pollutants, pesticides, and toxins. Specific non-limitingexamples include cAMP, cGMP, 8-bromoadenosine 3′,5′-cyclicmonophosphate, cholesterol, a hydroxycholesterol, vitamin D, 25-hydroxyvitamin D, vitamin B12, vitamin E, vitamin B1, vitamin B6, ascorbicacid, retinol, biotin, folate, legumin, salbutamol, melamine,sulfaquinoxaline, an inositol phosphate, a phosphatidyl inositolphosphate, prednisone, pregnenolone, dexamethasone, triamcinolone,fludrocortisone, dihydrotachysterol, oxandrolone, testosterone,dihydrotestosterone, nandrolone, norethindrone, medroxyprogesteroneacetate, progesterone, a glucocorticoid, aldosterone, estrogen,oxytocin, androstanediol glucuronide, bilirubin, warfarin, an estrogen,methotrexate, tobramycin, acetaminophen, encainide, fluoxetine,gentamycin, an aminoglycoside, amikacin, coenzyme Q10, theophylline,phenytoin, cimetidine, disulfiram, trazodone, ethanol, halothane,phenylbutazone, azapropazone, ibuprofen, amiodarone, imipramine,miconazole, metronidazole, nifedipine, chloramphenicol, trimethoprim, asulfonamide, rifampin, cisplatin, vinblastine, bleomycin, oxacillin,nitrofurantoin, phenobarbital, primidone, carbamazepine, metoclopramide,cholestyramine, colestipol, neomycin, sulfasalazine, digoxin,indomethacin, diltiazem, erythromycin, tetracycline, itraconazole,nicardipine, triamterene, spironolactone, chlorpromazine, a cyclosporin,nortriptyline, ethosuximide, imipramine, codeine, lorazepam, topiramate,disopyramide, levetiracetam, clobazam, oxcarbazepine, fructosamine,caffeine, methaqualone, meprobamate, fluphenazine, a barbiturate, aphenothiazine, serotonin, valproic acid, digitoxin, maprotiline,lidocaine, mexiletine, primidone, risperidone, OH-rispiridone, aporphyrin, haloperidol, flecainide, tocainide, acetazolamide, asulfonamide, verapamil, a metanephrine, an oxidized nucleotide, amycotoxin, tetrahydrocannabinol, a cannabinoid, cocaine, LSD, anamphetamine, a barbiturate, heroin, methadone, nicotine, cotinine, abenzodiazepine, bis(2-ethylhexyl) phthalate, bisphenol A, hydralazine,atrazine, an organochloride insecticide, an organophosphate insecticide,or a carbamate insecticide. In some embodiments, the analyte is asteroid hormone, a glucocorticoid, oxytocin, cAMP, cGMP,hydroxycholesterol, vitamin D or 25-hydroxy vitamin D.

The inventors have also discovered that multisignal labeling reagents,e.g., as described in U.S. Patent Publication 2011/0318788 and U.S. Pat.No. 7,514,551, are particularly effective whendigoxigenin/anti-digoxigenin is used as a binding pair to join thepolymer with the signal. See Examples 4-6.

Thus, in some embodiments, a multisignal labeling reagent is provided.The reagent comprises a first polymer covalently bound to (a) a reactivegroup or a first member of a first binding pair, and (b) more than onedigoxigenin molecule. Here, polymers, reactive groups and binding pairsare as described above.

In various embodiments, the digoxigenin molecules on the polymer areused as a binding pair member for joining a signal molecule, where theother member of the binding pair is an anti-digoxigenin antibody that isfurther bound to at least one signal molecule. Signal molecules aredescribed above, and could be, e.g., a fluorescent dye, a radioactivemolecule or an enzyme. In various embodiments, each signal comprises anenzyme that is alkaline phosphatase, horseradish peroxidase orluciferase.

The multisignal labeling reagent of these embodiments can furthercomprise a linker moiety covalently linking (a) the first polymer to thereactive group or first member of the first binding pair and/or (b) thefirst polymer to the more than one digoxigenin molecules. Linker groupshere are as described above.

In some embodiments, the first polymer is further bound to a secondpolymer, wherein the second polymer further comprises a signal moleculeor a first member of a second binding pair. In most constructs, thisconfiguration would cause a further amplification of the signal, e.g.,for use where high sensitivity is desired. In some of these embodiments,the first polymer is covalently bound to the second polymer.Alternatively, the first polymer is bound to the second polymer througha third binding pair.

In various aspects of these multisignal labeling reagents, the reagentis bound to a target molecule through the reactive group or the firstbinding pair. The target molecule is not narrowly limited to any classof compound. Examples include haptens, proteins, oligopeptides, nucleicacids, nucleic acid analogs, oligosaccharides, polysaccharides, lipidsand organic polymers.

Preferred embodiments are described in the following examples. Otherembodiments within the scope of the claims herein will be apparent toone skilled in the art from consideration of the specification orpractice of the invention as disclosed herein. It is intended that thespecification, together with the examples, be considered exemplary only,with the scope and spirit of the invention being indicated by theclaims, which follow the examples.

Example 1. Attaching an Oligonucleotide to cAMP (a) Synthesis of2′-O-Succinyl-cAMP-NHS Ester

2′-O-succinyl-cAMP (3.0 mg, 6.9 μmol) was dissolved in 650 μl ofanhydrous, amine-free DMSO and N-hydroxysuccinimide (11.5 mg, 100 μmol)was added. To the above clear solution,1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) (EDCI) (13.4 mg, 70μmol) dissolved in 150 μl of DMSO was added and the reaction mixture wasincubated at room temperature overnight. The reaction progress wasfollowed by TLC analysis (silica, isopropyl alcohol:NH₄OH:H₂O 6:3:1).The crude product was used as DMSO solution in the following step (b).

(b) Addition of Oligo to 2′-O-Succinyl-cAMP-NHS Ester

To a solution of 5amCap 22-mer (20 nmole, 5′-amino-C6-TTG CTG AGG TCATGG ATC GAG A-3′) in Modification Buffer (100 mM sodium phosphate, 150mM NaCl, pH 8.0), 45 molar equivalents of 2′-O-succinyl-cAMP-NHS esterin DMSO was added. The reaction mixture was incubated at roomtemperature for 18 h and after diluting with nuclease free water todecrease the amount of DMSO to 5% of the total, the mixture was purifiedon a 3 k Amicon diafiltration device. The cAMP-oligomer bioconjugate (˜8nmole) was identified and analyzed by HPLC analysis (Zorbax Oligocolumn, phosphate:acetonitrile gradient).

Example 2. Adding Poly Biotin to cAMP-Oligo

The cAMP-oligo described in Example 1 (0.6 nmole) was mixed with 0.9nmole of Targext99

(5′-TAT ATT ATA TTA TAT TAT ATT ATA TTA TAT TATATT ATA TTA TAT TAT ATT ATA TTA TAT TAT ATT ATATTA TAT TAT ATC TCG ATC CAT GAC CTC AGC-3′).The mixture contained 43 nmoles biotin-16-dUTP and 76 nmoles each ofdATP, dCTP and dGTP in 10 mM Tris-HCl, pH 7.9, 10 mM MgCl₂, 1 mM DTT and50 mM NaCl. T4 DNA polymerase exo minus (13 units) (Lucigen, Middleton,Wis.) was added, and the mixture was incubated at 37° C. for 90 min. Thereaction was stopped with the addition of EDTA to a final concentrationof 12.5 mM. The resulting poly-biotin cAMP was separated from freenucleotides using a NucAway (Ambion, Foster City, Calif.) size exclusionspin column equilibrated with phosphate buffered saline (PBS).

Example 3. Using cAMP Poly-Biotin to Quantify cAMP in a Sample

cAMP poly-biotin was used instead of cAMP-alkaline phosphatase conjugateto quantify low concentrations of cAMP in samples, in a competitiveELISA assay. Samples (100 μl), serial diluted standards, or a no cAMPcontrol were added to microtiter wells coated with goat-anti-rabbitantibody (Enzo Life Sciences). 50 μl of cAMP poly-biotin (10 pmol/ml)were added to the wells followed by an addition of 50 μl antibodysolution containing rabbit polyclonal antibody to cAMP (Enzo LifeSciences). Controls lacking rabbit anti-cAMP antibody were included. Theplate was sealed and mixed briefly on a plate shaker (˜500 rpm) for 5 sand incubated at 4° C. for greater than 12 hr. The contents of each wellwas emptied and washed three times, each with 400 μl wash buffer. Afterwashing, 200 μl of streptavidin-alkaline phosphatase (2 ng/ml) (ThermoScientific) was added to each well and incubated for 15 min on a plateshaker as above. The contents of the plate were emptied and wells washed4 times with wash buffer as before. Alkaline Phosphatase Yellow (pNPP)Liquid Substrate (Sigma) (100 μl) was then added to each well and theplate incubated at room temperature for 1 hour without shaking. Stopsolution consisting of 2 N NaOH (50 μl) was added to each well and theoptical density of each well was read at 405 nm using a plate reader.

The sensitivity of the assay, defined as the concentration of cAMPmeasured at 2 standard deviations from the mean of 16 control wellswithout added cAMP was determined to be between 0.03-0.06 pmol/ml, a5-10 fold increase over current assays using a cAMP-alkaline phosphataseconjugate. A sample result is shown in FIG. 1.

Example 4. Labeling a Biotinylated Oligonucleotide with MultipleDigoxigenin Molecules

The oligonucleotide 5′-biotin-triethylene glycol(TEG)-TTGCTGAGGTCATGGATCGAGA-3′ was extended with terminaldeoxynucleotidyl transferase as follows. The oligonucleotide (160pmoles) was mixed with 25 nmoles dATP, 5 (or 10) nmolesdigoxigenin-labeled dUTP (Roche Diagnostics, Indianapolis, Ind.), 1 mMcobalt chloride, 1×TdT reaction buffer (ENZO Life Sciences, Farmingdale,N.Y.) and 40 units of terminal deoxynucleotidyl transferase in a totalvolume of 25 μl. This was incubated at 30° C. for 2 h. The reaction wasstopped by the addition of EDTA to 12.75 mM. The extended oligo waspurified using a Nucaway spin column (Ambion, Austin, Tex.), followingthe manufacturer's instructions. Oligo dT₂₁ (800 pmoles) was added tobind to the poly dA tracts created using terminal transferase. A complexof streptavidin with the biotinylated digoxigenin labeledoligonucleotide was created by mixing 150 pmoles of oligo with 136pmoles streptavidin while vortexing.

Example 5. Labeling an Oligonucleotide Conjugated to Streptavidin withMultiple Digoxigenin Moieties

The oligonucleotide 5′-aminoC6-TTGCTGAGGTCATGGATCGAGA-3′ was attached tostreptavidin as described in Example 16 of U.S. Patent Publication2011/0318788. The streptavidin oligonucleotide was extended withterminal transferase as follows. The streptavidin oligonucleotide (180pmoles) was mixed with 25 nmoles dATP, 5 (or 10) nmoles digoxigeninlabeled dUTP (Roche Diagnostics, Indianapolis, Ind.), 1 mM cobaltchloride, 1×TdT reaction buffer (ENZO Life Sciences, Farmingdale, N.Y.)and 40 units of terminal deoxynucleotidyl transferase in a total volumeof 25 μl. This was incubated at 30° C. for a total of 2 hours. Thereaction was stopped by the addition of EDTA to 12.75 mM. The extendedoligo streptavidin was purified using a Nucaway spin column (Ambion,Austin, Tex.), following the manufacturer's instructions. 800 pmoles ofoligo dT21 was added to bind to the poly dA tracts created usingterminal transferase.

Example 6. Labeling an Oligonucleotide Conjugated to Streptavidin UsingDNA Polymerase and a Template

The oligonucleotide 5′-aminoC6-TTGCTGAGGTCATGGATCGAGA-3′ was attached tostreptavidin as described in Example 16 of U.S. Patent Publication2011/0318788. The streptavidin oligonucleotide was extended using atemplate oligonucleotide as follows. A second oligo (5′-ACTTCTACTTCTACTTCTAC TTCTACTTCT ACTTCTACTT CTACTTCTAC TCTTACTCTT ACTCTTCATTGGTCATCTCG ATCCATGACC TCAGC-3′) (MWG Operon, Huntsville, Ala.) washybridized to the oligo streptavidin. The streptavidin oligo construct(113 pmoles) was incubated with 338 pmoles of template oligo in 50 mMNaCl, 10 mM Tris-HCl, 10 mM MgCl₂, 1 mM dithiothreitol, pH 7.9, 6.1 nMolS4FB-dUTP, 21 nMol each of dATP, dCTP and dGTP and 5.4 units of T4 DNApolymerase exo⁻ (Lucigen, Middleton, Wis.) in a total volume of 30 at37° C. for 90 min. The extension reaction was stopped with 1 μl of 500mM EDTA, and the unincorporated nucleotides are removed using NucAwayspin columns (Applied Biosystems/Ambion, Austin, Tex.) as described bythe manufacturer (http://www.ambion.com/techlib/spec/sp_10070.pdf).

Example 7. Binding and Detecting Poly-Digoxigenin Labeled Streptavidin

The streptavidin-poly-digoxigenin from Example 5 and astreptavidin-alkaline phosphatase complex were tested by binding tobiotin attached to a 96-well microplate. Individual wells in themicroplate were coated with varying concentrations ofbiotinylated-bovine serum albumin (BSA). Biotinylated BSA was producedby mixing 0.75 μmol BSA (fraction V, Sigma-Aldrich, St. Louis, Mo.) with3.75 μmol ENZOTIN (NHS ester of biotin, ENZO Life Sciences, Farmingdale,N.Y.) in 50 mM sodium tetraborate, pH 8.5 at 22° C. for 4 hours.Unreacted biotin was removed using a Zeba spin column (Thermo-Pierce,Rockford, Ill.) as described by the manufacturer. The biotinylated-BSA(250 pg, 83.3 pg, 27.8 pg and 0 pg) was added to separate wells of blackMaxisorb (Nunc, Roskilde, Denmark) 96-well microplate, diluted in PBS.After 12 hours at 4° C., the excess biotin BSA was washed out of theplate using 3 washes of 0.05% Tween20® in PBS for 5 minutes each.Blocking buffer (Thermo-Pierce, Rockford, Ill.) containing 0.05%Tween20® and 50 μg/ml single-stranded salmon sperm DNA was added to eachwell and the plate was incubated at 22° C. for 15 minutes with shaking.The blocking buffer was then removed, and the streptavidin-polydigoxigenin or commercial streptavidin alkaline phosphatase (LifeTechnologies, Carlsbad, Calif.) was then added to each well (100 μl, 4nM) in the same blocking buffer and incubated with shaking for 60minutes at 22° C. After 60 minutes, the streptavidin complexes wereremoved from the digoxigenin wells, and those wells were washed oncewith 200 μl PBS with 0.05% Tween20®. After removal of the wash, 100 μlof alkaline phosphatase labeled digoxigenin antibody (Roche,Indianapolis, Ind.) diluted 750 fold in blocking buffer was added tothose wells. Incubation was continued at 22° C. for 60 minutes. Allliquid was removed, and each well was washed 3 times with 0.05% Tween20®in PBS, for 5 min each. CDP-Star (Life Technologies, Carlsbad, Calif.)(100 μl) was added to each well, and the chemiluminescence produced wasquantified using a BioTek SynergyMX (Winooski, Vt.) plate reader. Theresults are shown in FIG. 2.

All of the complexes with digoxigenin improved the signal compared tocommercial alkaline phosphatase-labeled streptavidin. Theoligonucleotide was more efficiently extended with terminal transferaseif it was not attached to streptavidin first. Extension with T4 DNApolymerase on the streptavidin-oligonucleotide complex appears to beefficient.

REFERENCES

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In view of the above, it will be seen that several objectives of theinvention are achieved and other advantages attained.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

All references cited in this specification are hereby incorporated byreference. The discussion of the references herein is intended merely tosummarize the assertions made by the authors and no admission is madethat any reference constitutes prior art. Applicants reserve the rightto challenge the accuracy and pertinence of the cited references.

What is claimed is:
 1. A method for providing an oligonucleotide labeledat each of the 5′ end and the 3′ end of the oligonucleotide, comprisingthe steps of: providing an DNA oligonucleotide labeled with a firstmember of a first binding pair at its 5′ end; adding deoxynucleotideslabeled with a first member of a second binding pair to the 3′ end ofthe oligonucleotide using terminal transferase.
 2. The method of claim1, wherein the first member of the first binding pair and the firstmember of the second binding pair are the same.
 3. The method of claim1, wherein the first member of the first binding pair and the firstmember of the second binding pair are different.
 4. The method of claim1, wherein the first member of the first binding pair is streptavidin oravidin.
 5. The method of claim 1, therein the first member of the secondbinding pair is digoxigenin.
 6. The method of claim 4, wherein the firstmember of the second binding pair is digoxigenin.
 7. A DNAoligonucleotide having a 5′ end and a 3′ end, comprising: a first memberof a first binding pair attached at the 5′ end of the DNAoligonucleotide; and a first member of a second binding pair attached atthe 3′ end of the DNA oligonucleotide.
 8. The DNA oligonucleotide ofclaim 7, wherein the first member of the first binding pair is aprotein.
 9. The DNA oligonucleotide of claim 8, wherein the first memberof the first binding pair is streptavidin or avidin.
 10. The DNAoligonucleotide of claim 7, wherein the first member of the secondbinding pair is digoxigenin.
 11. The DNA oligonucleotide of claim 8,wherein the first member of the second binding pair is digoxigenin. 12.The DNA oligonucleotide of claim 9, wherein the first member of thesecond binding pair is digoxigenin.